DESCRIPTION: Protein kinases phosphorylate intracellular substrates to regulate cell functions such as gene transcription. Kinase crystal structures indicate that they share similar structures and catalytic mechanisms. The kinase C-subunit of cAMP-dependent protein kinase (PKA) is used as a kinase model. This project proposes to utilize two natural catalytic (C) subunit variants, C-alpha and C-gamma and chimeric mutants of them, to identify structures that specify the regulation of gene transcription. C-alpha and C-gamma are highly homologous (83 percent identity), yet functionally distinct isozymes, thereby providing targets for structure-function differences. Different cAMP-mediated phenotypes are observed in C-gamma- versus C-alpha-expressing stable Kin8 clones. In transiently co-transfected cells, the isozymes exhibit remarkable differences in the regulation of CRE-reporter gene activity. In vitro, the isozymes exhibit differences in kinetics, substrate, and pseudosubstrate specificities. It is hypothesized that the isozyme-specific phenotypes are due to differences between C-alpha and C-gamma in the recognition of substrates and/or pseudosubstrates. The specific aims are: (1) to utilize native and mutant C-gamma and C-alpha isozymes and differences in CRE-reporter gene activity to define kinase structures that determine the regulation of CRE-gene transcription in intact cells; (2) to utilize recombinant C-gamma and C-alpha subunits in vitro to define differences in the isozymes for the recognition of substrates and pseudosubstrate inhibitors; and (3) to determine the specificities of mutants that differ for CRE-gene expression in intact cells.